Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (Mtb) is one of the world's leading public health concerns, responsible for the deaths of more than 2 million people annually. The concurrent HIV epidemic coupled with the emergence of extensively drug resistant strains of Mtb have greatly amplified the impact of M. tuberculosis infection and created a global healthcare crisis. One of the problems facing those tasked with treating tuberculosis is that even infections caused by drug susceptible Mtb strains require long-term treatment in part because the anti-mycobacterial agents used currently are inefficient at killing host adapted bacteria. The primary objective of this work is to identify Mtb genes essential for adaptation to survival in the lung particularly those not identified in earlier screening approaches. The premise is that the more known about the physiology of the organism during infection, the greater is the opportunity to design successful therapeutic interventions which target in vivo essential bacterial enzymes and pathways. This work employs a whole genome screen of an Mtb transposon mutant library administered via aerosol to guinea pigs. The guinea pig host reproduces many aspects of human tuberculosis including the necrotic, hypoxic granuloma not found in all model systems, for example the mouse. The proposal has two AIMS. AIM1 is to apply Transposon Capture and Sequencing or TraCS analysis to existing guinea pig infection derived libraries. TraCs is a robust and highly sensitive deep sequencing based methodology, which allows the detection of mutations in Mtb genes which render the bacilli incapable of survival in the pulmonary environment. The second AIM is a confirmatory analysis of the screen wherein individual deletion alleles will be constructed in two genes identified in the original screen. The in vivo phenotype of the deletion mutants will be examined in the low dose aerosol guinea pig and murine tuberculosis infection models, and results will be compared to those observed with infection of wild type and complemented mutant strains. The inclusion of a murine infection cohort allows interspecies comparison and validation of the potential exclusivity of the particular gene for maintenance of disease in the human-like pulmonary environment modeled by the guinea pig. Ultimately this work will contribute to the development of therapeutics which target pathways essential for bacilli survival in the host environment.